Horizontal cells capable of selectively accumulating indoleamines have been found in the retina of Saimiri sciureus, the New World squirrel monkey. The uptake of 3H-serotonin (5HT) in vivo and in vitro has been localized by light and electron microscopic (EM) autoradiography only to horizontal cells. In the central retina up to 50% and in the periphery 15-20% of the somas lying at the scleral border of the inner nuclear layer are labeled. In EM autoradiographs, labeled processes occur as lateral elements in both cone and rod synaptic invaginations, but grain counts show four times as many rods as cones are labeled. The light-dark adaptational state of the retina has no apparent effect on 3H-5HT uptake, but it is temperature dependent, and is saturable as indicated by its inhibition in the presence of an excess of nonradioactive 5HT. The indoleamine-accumulating capacity of this population of horizontal cells also was demonstrated by histofluorescence microscopy. A single row of fluorescent cells with a round soma was found lying at the outer edge of the inner nuclear layer. These cells send fluorescent processes only into the outer plexiform layer. The authors suggest that 5HT is not the native transmitter of these squirrel monkey horizontal cells because they lack intrinsic fluorescence and only could be visualized after uptake of extrinsically applied indoleamines. The true transmitter is likely to be a closely related but nonfluorescent indole.